U.S. patent application number 10/515253 was filed with the patent office on 2005-06-09 for radio lan access authentication system.
Invention is credited to Honda, Mitsuo, Izumi, Yasuyuki, Matsuo, Kazuhiro.
Application Number | 20050123561 10/515253 |
Document ID | / |
Family ID | 29545071 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050123561 |
Kind Code |
A1 |
Honda, Mitsuo ; et
al. |
June 9, 2005 |
Radio lan access authentication system
Abstract
This invention provides a recombinant BCG vaccine transformed by
an expression vector having polynucleotide coding for exogenous
antigenic protein, characterized in that, the BCG vaccine is used
for initial antigen stimulus in immune induction by plural antigen
stimulations and also provides a method for the immune induction
where the initial antigen stimulus is carried out by the said BCG
vaccine and one or more additional antigenic stimulation(s) is/are
carried out by non-BCG vaccine expressing the same antigenic
protein.
Inventors: |
Honda, Mitsuo; (Tokyo,
JP) ; Matsuo, Kazuhiro; (Kanagawa, JP) ;
Izumi, Yasuyuki; (Tokyo, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
29545071 |
Appl. No.: |
10/515253 |
Filed: |
January 18, 2005 |
PCT Filed: |
November 20, 2002 |
PCT NO: |
PCT/JP02/12125 |
Current U.S.
Class: |
424/199.1 |
Current CPC
Class: |
A61K 2039/523 20130101;
C12N 2740/15022 20130101; A61K 2039/545 20130101; A61P 31/18
20180101; A61P 31/00 20180101; C07K 14/005 20130101; A61K 39/0011
20130101; A61P 37/02 20180101 |
Class at
Publication: |
424/199.1 |
International
Class: |
A61K 039/12 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2002 |
JP |
2002-145132 |
Claims
1. A recombinant BCG vaccine transformed with an expression vector
having a polynucleotide encoding an exogenous antigenic protein,
which the BCG vaccine is used for initial antigen stimulation in
immune induction by plural antigen stimulations.
2. The BCG vaccine according to claim 1, wherein the antigenic
protein is derived from immunodeficiency virus.
3. The BCG vaccine according to claim 2, wherein the antigenic
protein of immunodeficiency virus is a product of an HIV gene.
4. A method for the immune induction by plural stimulations of
exogenous antigenic protein, which comprises carrying out the
initial antigen stimulation by the BCG vaccine of claim 1, and
carrying out one or more additional antigenic stimulations by
non-BCG vaccine expressing the same antigenic protein.
5. The method according to claim 4, wherein the vaccine for the
additional antigenic stimulations is a recombinant vaccinia virus
vaccine.
6. The method according to claim 4, wherein the antigenic protein
is derived from immunodeficiency virus.
7. The method according to claim 6, wherein the antigenic protein
of the immunodeficiency virus is an HIV gene product.
Description
TECHNICAL FIELD
[0001] The invention of this application relates to BCG vaccine and
utilization thereof. More particularly, the invention of this
application relates to a recombinant BCG vaccine used for initial
antigen stimulation in immune induction for prevention and
treatment of various infectious diseases, cancer, etc. and to a
method for induction of immunity in human beings or animals using
the BCG vaccine.
BACKGROUND ART
[0002] Attenuated BCG strain of Mycobacterium bovis (hereinafter,
referred to as "BCG") is well known and is the most commonly used
live bacterial vaccine due to its safety.
[0003] On the other hand, as a result of development and
improvement in genetic recombination technique in the past two
decades, there have been vigorous studies where microbes such as
virus and bacteria are modified so that they express exogenous
antigenic protein and are applied as vaccine vector for prevention
and therapy of various infectious diseases and cancer. With regard
to BCG, there have been reported recombinant BCG vaccines where
human immunodeficiency virus (HIV) or simian immunodeficiency virus
(SIV) is the target (J. Immunol. 164:4968-4978, 2000; J. Virol.
71:2303-2309, 1997; Infect. Immun. 57:283-288, 1989). Vaccine where
HIV gene is expressed in BCG is able to induce immunity for a long
period (at least for two years) and this immunity-inducing ability
for such a long period is an excellent characteristic which is not
noted in other DNA vaccines.
[0004] However, in the case of the conventional recombinant BCG
vaccines, they are not always sufficient ability to induce immunity
to the infectious disease, cancer, et al which is the target. For
example, when immune induction is carried out to guinea pigs using
a recombinant BCG vaccine using HIV-1 as a target, it is necessary
to administer the dose which is from 50 to 100-fold of the usual
dose (0.05-0.1 mg) of conventional BCG vaccine to human beings
(Proc. Natl. Acad. Sci. USA 92:10698-10697, 1995). Further, in a
test using macaque models, administration of only recombinant BCG
for prevention from infection of pathogenic virus did not give
favorable results.
[0005] Recombinant BCG vaccine is an excellent candidate for
vaccine in view of its duration of effective immunity, safety and
ease of production, and its effective utilization is urgently
needed in the medical field.
[0006] The invention of this application has been achieved in view
of the above-mentioned circumstances and has an object of providing
a novel means for an effective utilization of recombinant BCG
vaccine.
DISCLOSURE OF THE INVENTION
[0007] As the first invention for solving the above-mentioned
problems, this application provides a recombinant BCG vaccine
transformed with an expression vector having a polynucleotide
encoding an exogenous antigenic protein, which the BCG vaccine is
used for initial antigen stimulation in immune induction by plural
antigen stimulations.
[0008] As the second invention, this application provides a method
for the immune induction by plural stimulations of exogenous
antigenic protein, which comprises carrying out the initial antigen
stimulation by the BCG vaccine of claim 1, and carrying out one or
more additional antigenic stimulations by non-BCG vaccine
expressing the same antigenic protein.
[0009] In the method of the second invention, it is a preferred
embodiment that the vaccine for additional antigenic stimulation is
a recombinant vaccinia virus vaccine such as recombinant DIs
vaccine.
[0010] In addition, in the first and second inventions, it is a
preferred embodiment that the antigenic protein is derived from
immunodeficient virus or, to be more specific, the antigenic
protein of immunodeficient virus is an HIV gene product such as
Gag.
[0011] Thus, in accordance with the method of this invention, the
virus acquired by a single administration of, for example,
recombinant vaccinia virus (such as recombinant DIs-gag vaccine)
can be prevented almost completely from flowing out into the blood
and a decrease in CD4 cells can be suppressed as well. As a result,
it is now possible to suppress the spread of at least pathogenic
virus in the body and to prevent the progress of infectious
diseases.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a schematic chart exemplifying the constitution of
expression vector pSO-SIVgag used for the preparation of the
recombinant BCG strain (rBCG-SIVgag) in Example 1.
[0013] FIG. 2 is the result of a Western blot analysis where amount
of Gag protein produced from rBCG-SIVgag was measured.
[0014] FIG. 3 shows the change with the lapse of time of the number
of copies of viral RNA (left drawing) and the change of CD4 cell
counts with the lapse of time (right drawing) in blood of a control
macaque when infected with pathogenic virus.
[0015] FIG. 4 shows the change with the lapse of time of the number
of copies of viral RNA (left drawing) and the change with the lapse
of time of CD4 cell counts (right drawing) in blood after infection
with pathogenic virus in a macaque where rBCG-SIVgag only was
vaccine-inoculated one time.
[0016] FIG. 5 shows the change with the lapse of time of the number
of copies of viral RNA (left drawing) and the change with the lapse
of time of CD4 cell counts (right drawing) in blood after infection
with pathogenic virus in a macaque where rDIs-SIVgag only was
vaccine-inoculated one time.
[0017] FIG. 6 shows the change with the lapse of time of the number
of copies of viral RNA (left drawing) and the change with the lapse
of time of CD4 cell counts (right drawing) in blood after infection
with pathogenic virus in a macaque where rDIs-SIVgag+rBCG-SIVgag
was vaccine-inoculated.
[0018] FIG. 7 shows the change with the lapse of time of the number
of copies of viral RNA in blood after infection with pathogenic
virus in a macaque where rBCG-SIVgag+rDIs-SIVgag was
vaccine-inoculated. FIG. 7/1 shows the change with the lapse of
time of CD4 cell count in blood after infection with pathogenic
virus in a macaque where rBCG-SIVgag+rDIs-SIVgag was
vaccine-inoculated.
[0019] FIG. 8 shows the change with the lapse of time of the number
of copies of viral RNA in blood after inoculation with control
vaccine (vector) to a macaque having a BCG anamnestic reaction.
FIG. 8/1 shows the change with the lapse of time of CD4 cell count
after inoculation with control vaccine (vector) to a macaque having
a BCG anamnestic reaction.
[0020] FIG. 9 shows the change with the lapse of time of the number
of copies of viral RNA in blood after vaccine-inoculation of
rBCG-SIVgag (oral)+rDIs-SIVgag (intravenous) to a macaque having a
BCG anamnestic reaction. FIG. 9/1 shows the change with the lapse
of time of CD4 cell count after vaccine-inoculation of rBCG-SIVgag
(oral)+rDIs-SIVgag (intravenous) to a macaque having a BCG
anamnestic reaction.
[0021] FIG. 10 shows the change with the lapse of time the number
of copies of viral RNA in blood after vaccine-inoculation with
rBCG-SIVgag (intravenous)+rDIs-SIVgag (intravenous) to a macaque
having a BCG anamnestic reaction. FIG. 10/1 shows the change with
the lapse of time of CD4 cell count in blood after
vaccine-inoculation with rBCG-SIVgag (intravenous)+rDIs-SIVgag
(intravenous) to a macaque having a BCG anamnestic reaction.
BEST MODE FOR CARRYING OUT THE INVENTION
[0022] The first invention is a recombinant BCG vaccine which is
transformed by an expression vector having polynucleotide encoding
exogenous antigenic protein. In immune induction by antigenic
stimulation plural times, this recombinant BCG vaccine is
characterized in that its use is for the initial antigen
stimulation. Thus, the inventors of this application have found
that, even in the case of recombinant BCG vaccine having
insufficient immunity-inducing ability when only it is used, it
enhances a specific immunity when it is used as a initial antigen
stimulus (priming) followed by additional antigen stimuli
(boosting), whereupon the present invention has been achieved.
[0023] With regard to the BCG strain, it is possible to use widely
known ones which have been used for vaccination of tuberculosis,
etc. With regard to the expression vector, it is possible to use a
vector for BCG (such as plasmid pSO246) which has been used for the
preparation of conventional recombinant BCG vaccine. When
polynucleotide coding for the desired antigenic protein which is
exogenous (in other words, not one of BCG) is inserted into a
cloning site of this vector, it is possible to construct the
expression vector. Incidentally, in the following description,
exogenous antigenic protein may be referred to as "exogenous
polypeptide" while polynucleotide coding for it may be referred to
as "exogenous polynucleotide". In addition, any promoter and
terminator sequences derived from BCG strain (such as promoter and
terminator sequences of heat shock protein (HSP) derived from BCG)
are ligated to the polynucleotide, whereupon the exogenous
polypeptide is well expressed.
[0024] An exogenous polynucleotide is a polynucleotide (such as
cDNA fragment) which codes for antigenic protein other than one of
a BCG strain. Anything may be used as the exogenous polypeptide as
long as it brings about an antigen-antibody reaction in vivo. To be
more specific, gag precursor p55 or p24 protein, env protein gp120
or gp160, pol precursor protein, nef protein, tat protein, etc.
which are proteins of human immunodeficiency virus (HIV) which is a
virus causing human acquired immune deficiency syndrome (AIDS) may
be used as objects. It is also possible to use a similar antigenic
polypeptide derived from simian immunodeficiency virus (SIV). It is
further possible to use a polynucleotide coding for antigenic
protein of cancer cells or other pathogens (other pathogenic
viruses and bacteria).
[0025] With regard to a method for obtaining the exogenous
polynucleotide, the significant sequence of the polynucleotide is
cut out by an appropriate restriction enzyme from genome gene
coding for exogenous polypeptide or cloned plasmid cDNA, or it is
amplified by a polymerase chain reaction (PCR) using primer of an
appropriate sequence. When it is not cloned, it is possible to
obtain the above by amplification of DNA fragment by means of the
above PCR using genomic DNA of animals or cells having that gene
or, in the case of virus, using DNA or RNA derived from animal
cells infected with virus as a template.
[0026] The expression vector constructed as such is introduced into
BCG strain by known methods such as a calcium chloride method or an
electroporation method and expression of the exogenous polypeptide
of a transgenic microorganism is confirmed by a western blotting or
by known immunological measuring method (such as ELISA) whereby the
recombinant BCG of this invention can be prepared.
[0027] When the recombinant BCG thus prepared is suspended in a
liquid carrier which is similar to that in the case of usual BCG
vaccine, a recombinant BCG vaccine can be prepared and the
resulting vaccine is able to be actually used for an immune
induction method of the second invention.
[0028] A method of the second invention is characterized in that
the initial antigen stimulus is carried out by the BCG vaccine of
the above-mentioned first invention and one or more additional
antigenic stimulation(s) is/are carried out by non-BCG vaccine
expressing the same antigenic protein.
[0029] Vaccine for the additional antigenic stimulation (booster
vaccine) can be prepared by transformation of known viruses or
bacteria used for recombinant vaccine (such as poliovirus,
influenza virus, rhinovirus, varicella virus, vaccinia virus,
Salmonella bacteria and Listeria bacteria) with the same exogenous
polynucleotide as the recombinant BCG vaccine (prime vaccine) of
the first invention. In a method of the invention, a recombinant
vaccinia virus DIs vaccine which was previously developed (Japanese
Patent Laid-Open No. 20002/017370) by the inventors of this
application is a preferred booster vaccine.
[0030] Administration of prime vaccine and booster vaccine can be
carried out by known methods such as injection or oral
administration. Although the dose and the schedule may be different
depending upon type (human being or animal), body weight, type of
the immunity to be induced, etc. of the individual to be inspected,
prime vaccine may be 0.01 to 10 mg and booster vaccine may be
10.sup.5 to 10.sup.10 PFU for example. The time interval between
inoculations of vaccine may be 3 to 12 months.
[0031] The invention of this application will now be illustrated as
hereunder in more detail and specifically by way of the following
Examples although the invention of this application is not limited
by the following examples.
EXAMPLES
Example 1
Preparation of Recombinant BCG
[0032] SIV gag gene was isolated from a plasmid pNL432 (J. Virol.
59:284-291, 1986), hsp60 promoter derived from BCG strain and
terminator were ligated to front and rear of the said gene DNA,
respectively and that is inserted into a multicloning site of a
shuttle vector pSO246 of Escherichia coli-BCG strain (FEMS
Microbiol. Lett. 135:237-243, 1996) whereupon an expression vector
pSO-SIVgag was constructed (FIG. 1).
[0033] The expression vector was introduced into BCG Tokyo strain
using Gene-pulser (Bio-Rad) according to a published method (Proc.
Natl. Acad. Sci. USA 85:6987-6991, 1988) and the transformant was
selected on a Middlebrook 7H10 agar medium (Difco) containing 20
.mu.g/ml of kanamycin to prepare a recombinant BCG strain
(rBCG-SIVgag) having pSO-SIVgag.
[0034] As a result of confirmation of production of SIVgag protein
by a Western blotting, a 55 kDa protein was detected in an extract
of rBCG-SIVgag as shown in FIG. 2. On the contrary, no Gag protein
was detected in rBCG-pSO246 which was a control. Concentration of
the SIV Gag protein was 45.+-.12 ng per mg of rBCG-SIVgag and such
a productivity level was maintained during at least 450 passages in
vitro.
Example 2
Immune Induction
[0035] Immune induction was carried out in cynomolgus monkey using
the recombinant BCG strain prepared in Example 1 (rBCG-SIVgag) and
a recombinant vaccinia DIs (rDIs-SIVgag). Incidentally, the
rDIs-SIVgag was prepared by the same method mentioned in Example 1
of Japanese Patent Laid-Open No. 2002/017370 using SIVgag instead
of HIV-1gag in the said patent.
[0036] Fourteen cynomolgus monkeys were divided into the following
five groups and immunization (boosting) was carried out at the
stage of 0, 47 and 54 week(s) after the initial antigen
stimulus.
[0037] Group 1 (four macaques): control (one was a nave macaque
while the other three were intracutaneously inoculated with
rBCG-pSO246 once and intravenously inoculated with rDIs-LacZ
(10.sup.6 PFU) twice)
[0038] Group 2 (two macaques): Intracutaneously inoculated with
rBCG-SIVgag (10 mg) once
[0039] Group 3 (two macaques): Intravenously inoculated with
rDIs-SIVgag (10.sup.6 PFU) twice
[0040] Group 4 (three macaques): Intravenously inoculated with
rDIs-SIVgag (10.sup.6 PFU) twice and intracutaneously inoculated
with rBCG-SIVgag (10 mg) once
[0041] Group 5 (three macaques): Intracutaneously inoculated with
rBCG-pSO246 (10 mg) once and intravenously inoculated with
rDIs-LacZ (10.sup.6 PFU) twice
[0042] Then, ten weeks after the second immunity booster, the
macaques were challenged with pathogenic virus (SHIV-KS661; 2000
TCID.sub.50) via mucous membrane intrarectally, and the change in
the number of virus copies and CD4 cell counts in blood were
measured periodically.
[0043] When the macaques of the control group were challenged with
SHIV-KS661, CD4 decreased to {fraction (1/10)} to {fraction
(1/100)} of original levels after about two weeks and, on the other
hand, the number of copies of viral RNA in the blood increased by
10.sup.8-9, quickly arrived at a set point and shifted to a level
of 10.sup.5-6. In the case of rDIs-SIVgag only (group 3; FIG. 5),
changes in CD4 counts and viral RNA numbers were also as same as
those in the case of control. Further, in the case of macaques
treated with rDIs-SIVgag (initial antigen stimulus)+rBCG-SIVgag
(boosting) (group 4), there was noted the same changes with the
lapse of time as in the case of the control and the single
immunization.
[0044] On the contrary, in the case of macaques of group 5
(rBCG-SIVgag+rDIs-SIVgag), the number of copies of viral RNA
significantly decreased, indicating that a strong Gag-specific
immunity was induced as shown in FIG. 7. Decrease in CD4 cells was
also significantly suppressed.
[0045] From the above results, it was confirmed that a strong
specific immunity was induced when a initial antigen stimulus was
conducted using a recombinant BCG vaccine (rBCG-SIVgag) and
boosting was carried out using a recombinant DIs vaccine
(rDIs-SIVgag).
Example 3
Suppression of Anamnestic Reaction of BCG
[0046] Influence of the immune induction method of this invention
on BCG anamnestic reaction was investigated. Specifically, to
simulate influence of administration in a human being, BCG Tokyo
strain (0.1 mg) was inoculated to cynomolgus monkeys about two
years before and, after confirming that DTH was clearly induced
even after two years, vaccine was inoculated to the macaques of the
following three groups.
[0047] Group 1 (3 macaques): Oral inoculation of rBCG-pSO246 (80
mg) twice+intravenous inoculation of rDIs-LacZ (10.sup.6 PFU)
twice
[0048] Group 2 (2 macaques): Oral inoculation of rBCG-SIVgag (80
mg) twice+intravenous inoculation of rDIs-SIVgag (10.sup.6 PFU)
twice
[0049] Group 3 (1 macaques): Intravenous inoculation of rBCG-SIVgag
(10 mg) once+intravenous inoculation of rDIs-SIVgag (10.sup.6 PFU)
once
[0050] The two BCG priming inoculations were carried out at the
stage of -48 and 0 week(s) and two boostings were carried out after
27 and 57 weeks. In the group 3, the initial antigen stimulus was
conducted at the stage of 0 week and a boosting was conducted at
the stage of 57 weeks.
[0051] After about three months from the final vaccine inoculation,
the macaque was challenged with pathogenic virus (SHIV-C2/1
20TCID.sub.50) and the change in CD4 cell counts and viral RNA copy
numbers were measured periodically.
[0052] The results are as shown in FIGS. 8 through 10. Changes with
the lapse of time of viral RNA numbers and CD4 counts (FIG. 10) by
rBCG-SIVgag initial antigen stimulus (intravenous)+rDIs-SIVgag
boosting (intravenous) (group 3) were as same as those in the
control (group 1). On the contrary, in the case of rBCG-SIVgag
initial antigen stimulus (oral) and rDIs-SIVgag boosting
(intravenous) (group 2), there were noted a significant decrease in
viral amount in blood and a suppression of decrease in CD4
cells.
[0053] From the above results, it was confirmed that, when initial
antigen stimulus of rBCG-SIVgag was carried out by oral
administration and boosting of rDIs-SIVgag was carried out by
intravenous inoculation, influence on anamnestic reaction could be
excluded not only regarding immune induction but also in protective
immunity.
Industrial Applicability
[0054] As fully illustrated hereinabove, an effective immune
induction using a recombinant BCG vaccine is now possible in
accordance with the invention of this application and an effective
prevention of various infectious diseases, cancers, etc. can be
achieved.
* * * * *